CD14 , TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

Toll‐like receptor 4 ( TLR4 ) is responsible for the immediate response to Gram‐negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal‐ MyD88 [Mal (MyD88‐adaptor‐like) ‐ MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro‐inflammatory cytokines while TRAM‐TRIF [TRAM (TRIF‐related adaptor molecule)‐TRIF (TIR‐domain‐containing adapter‐inducing interferon‐β)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide ( LPS ) stimulation. We found that LPS induced a CD14 ‐dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy ( TIRF ) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14 / LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373‐CD14 . These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment ( ERC ) and to early sorting endosomes. Moreover, CD14 / LPS but not TRAM was immobilized on RAB11A ‐positive endosomes, which indicates that TRAM and CD14 / LPS can independently be recruited to endosomes.